CN109912691A - A kind of preparation method of (5-oxo-D-Proline)-leuprorelin acetate - Google Patents

A kind of preparation method of (5-oxo-D-Proline)-leuprorelin acetate Download PDF

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CN109912691A
CN109912691A CN201910094532.9A CN201910094532A CN109912691A CN 109912691 A CN109912691 A CN 109912691A CN 201910094532 A CN201910094532 A CN 201910094532A CN 109912691 A CN109912691 A CN 109912691A
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connect
peptide
preparation
nonapeptide
dipeptides
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王林鹏
张道桂
单连民
万龙岩
江晓漫
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SHANGHAI LIZHU PHARMACEUTICAL CO Ltd
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SHANGHAI LIZHU PHARMACEUTICAL CO Ltd
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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Abstract

The present invention, which provides, a kind of prepares (5-oxo-D-Proline)] method of-leuprorelin acetate; include: that (1) solid phase connects peptide: passing through the step of preparing raw material, connecing dipeptides, connect tripeptides, connect tetrapeptide, connect pentapeptide, connect hexapeptide, connect heptapeptide, connect octapeptide and connect nonapeptide; obtain Leuprorelin nonapeptide resin; wherein, connect dipeptides to include the step of connecing nonapeptide deprotection base；It then washs, neutralizes, washs again, connecing peptide and detection free ammonia；(2) crude product aminolysis: is dissociated from above-mentioned Leuprorelin nonapeptide resin by ethylamine；(3) HPLC is purified: obtaining leuprorelin acetate intermediate purification product through HPLC preparation column purification；(4) it is concentrated；(5) it filters, be lyophilized.

Description

A kind of preparation method of (5-oxo-D-Proline)-leuprorelin acetate
Technical field
The present invention relates to a kind of chemical synthesis process, and in particular to preparation (5-oxo-D-Proline)-leuprorelin acetate Preparation method.
Background technique
Leuprorelin acetate production of raw medicine uses preparation Boc-Pro-- solid phase to connect peptide-aminolysis-HPLC purifying-dense at present Contracting-filtering lyophilized technique connects peptide-acid cutting-ethylamine modification-side chain full guard cutting-using preparation FMoc-Pro-- solid phase HPLC purifying-concentration-filtering lyophilized technique.Wherein, solid phase connect peptide be by prepare raw material, connect dipeptides, connect tripeptides, connect tetrapeptide, The step of connecing pentapeptide, connecing hexapeptide, connect heptapeptide, connecing octapeptide and connect nonapeptide, obtains Leuprorelin nonapeptide resin.
During solid phase connects peptide, since the side reactions such as racemization occur, a variety of by-products can be generated, although by purifying By-product is removed, but still has part and remains in final finished product bulk pharmaceutical chemicals.European Union's pharmacopeia (Japan and United States Pharmacopeia It is described) know that remaining in known related substance in bulk pharmaceutical chemicals mainly includes following 11 kinds: [3-D- tryptophan]-acetic acid is bright Third Rayleigh, [2-D- histidine, 4-D- serine]-leuprorelin acetate, [7-D- leucine]-leuprorelin acetate, [4-D- Propylhomoserin]-leuprorelin acetate, [2-D- histidine]-leuprorelin acetate, (5-oxo-D-Proline)]-acetic acid bright third is auspicious Woods, [8- [5-N- [imino (1H-pyrazol-lyl)-L-ornithine]]-leuprorelin acetate, [6-L- leucine]-vinegar Sour Leuprorelin, [5-D- tyrosine]-leuprorelin acetate, [4-dehydroalanine]-leuprorelin acetate, [4- (0- Acetyl-L- serine)]-leuprorelin acetate.Wherein, (5-oxo-D-Proline)]-leuprorelin acetate structural formula is such as Under: D- pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-ProNHC2H5。
It is above-mentioned it is each in relation to substance other than the content in finished product must be strictly controlled, physicochemical property, whether Adverse reaction and toxicological effect etc. are generated to patient, also required as clear as possible.To be carried out to above-mentioned each related substance Further investigation, it is necessary to obtain sufficient amount, structure correctly and the related substance of high-purity is just particularly important.
The above-mentioned approach in relation to substance is obtained at present predominantly when Dichlorodiphenyl Acetate Leuprorelin crude product is purified, to micro Related substance be collected, related amount of substance is few obtained by this method and purity is lower.Either be collected into it is a certain amount of after, then to it It is purified to obtain, related material purity obtained by this method is higher, but needs to be enriched with multiple batches of crude product just and can obtain and is a certain amount of Related substance, the period is long, at high cost.Above by purification of target peptide, the method in relation to substance is obtained by crude product purity and pure The separating degree of change is affected, when in crude product in relation to substance it is less and when purifies and separates degree is lower (such as: opposite appearance time is 0.7 to 0.78 4 kinds of related substances, when purifying, are removed together, 3 kinds of related substances that opposite appearance time is 0.9 to 1.09 It is similar to target peak, it is difficult to separate), it is difficult to obtain the specific required related substance of structure.
There has been no synthesis (5-oxo-D-Proline) at present] relevant report of-leuprorelin acetate.
Summary of the invention
This patent is 0.94 to opposite appearance time according to European Union's pharmacopeia (Japan and United States Pharmacopeia are also described), English Letter is I[(5-oxo-D-Proline)]-leuprorelin acetate] in relation to substance, another method is provided, i.e., it is straight with synthetic method It is bonded into out crude product, the target product of high-purity is then obtained by HPLC purifying-concentration-filtering-vacuum freeze drying again.
This patent provides [(5-oxo-D-Proline)]-leuprorelin acetate] synthetic method in relation to substance.We Method is Boc method solid phase synthesis process, by the way that reaction temperature, different aminoacids use different activators, de- BOC protecting group The application of reagent, the degree of substitution of BOC-Pro is continuously improved in BOC-Pro- resin, not only overcomes the Fmoc method production cycle Long, at high cost disadvantage, biggest advantage of the present invention is to minimize side reaction, hence it is evident that improves BOC method crude product synthetic yield Reach 60% or more, while substantially increasing crude product quality, reduces production cost.
The preparation method of this patent, specifically:
1, solid phase connects peptide: by preparing raw material, connecing dipeptides, connect tripeptides, connect tetrapeptide, connect pentapeptide, connect hexapeptide, connect heptapeptide, connect Octapeptide and the step of connect nonapeptide, obtains Leuprorelin nonapeptide resin, wherein connects dipeptides to including deprotecting the step of connecing nonapeptide Base washing, neutralization, washs again, connects peptide and detection free ammonia；Connect in peptide, connect tetrapeptide and nonapeptide be D amino acid, other be L Amino acid；
Wherein, the preparation raw material include: byResin is with anhydrous CH2Cl2Washing, stirring, is drained；It is described 'sResin replaces equivalents to be 0.4-0.7mmol/g
2, crude product aminolysis: is dissociated from above-mentioned Leuprorelin nonapeptide resin by ethylamine
3, HPLC is purified: obtaining leuprorelin acetate intermediate purification product through HPLC preparation column purification；
4, it is concentrated；
5, it filters, be lyophilized；
And in step 1, dipeptides is connect to when connecing nonapeptide, it is 9 that the deprotection base, which includes: by the raw material addition concentration of preparation, HCl/iPrOH solution, the CH of~10N2Cl2, thioglycol three mixed liquor in, stirring, drain, wherein HCl/iPrOH is molten Liquid, CH2Cl2, thioglycol three volume ratio be 5: 4: 1.
D- amino acid uses efficient condensing agent HOAt and DlC, and l-amino acid uses HOBt and DCCI；When connecing peptide, first will The lysate of amino acid monomer and efficient condensing agent, pre-activate is carried out in ice bath, filters out the by-products such as N- acylureas again -5 DEG C to -22 DEG C, condensation reaction is carried out, side reaction is reduced.
In step 1, after each peptide reaction of 2 to 9 peptides, dehydrated alcohol and methanol washing is not used, washing is reduced In the process because resin shrinkage bring adversely affects.Gained nonapeptide resin is 0.5 hour dry or more not in 50 DEG C of baking ovens, keeps away Exempting from high temperature causes peptide to be broken.Acetone used carries out pre-freeze to -20 DEG C hereinafter, high temperature precipitations is avoided to cause peptide chain irreversible when aminolysis Reaction.
The preparation method of this patent, specially following steps:
1, it connects dipeptides: a) taking a certain amount ofResin (substitution equivalents is 0.55mmol/g), washing is taken out It is dry；
B) it deprotects base: HCl/iPrOH, CH2Cl2, mercaptoethanol is added, stirring is drained；
C) wash: CH2Cl2 washing is drained；DMF washing, is drained；
D) it neutralizes: triethylamine/CH is added2Cl2(10/90) it washs, drains；Washing: DMF washing is drained；CH2Cl2Washing To being in neutrality, drain；
E) peptide is connect: Boc-ArgHCl (H2O) 1
HOBt 1.0-1.15
DCCI 1.0-1.15
F) amino acid monomer；HOBt and DCCI, is dissolved with DMF respectively, first by the lysate of amino acid monomer and HOBt Lysate mixing, ice bath stirring react 30-60 minutes, then add DCCI lysate, continue to be stirred to react, and filter, gained Clear liquid pours into resin, close plug bottleneck, -5 DEG C to -22 DEG C reactions；Solvent is drained, with CH2Cl2Washing, DMF washing, CH2Cl2It washes It washs, drains.
2, connect tripeptides: deprotection base, washing, neutralization: method is same to be connect under dipeptides item.
Connect peptide: Boc-Leu (H2O) 1
HOBt 1.15(1.0-1.15)
DCCI 1.15 (1.0-1.15) method is same to be connect under dipeptides item,
3, connect tetrapeptide deprotection base, washing, neutralization: method is same to be connect under dipeptides item.
Connect peptide: Boc-D-Leu 1
HOAt 1.15(1.0-1.15)
DIC 1.15(1.0-1.15)
Method is same to be connect under dipeptides item,
4, connect pentapeptide: protecting group, washing, neutralization: method is same to be connect under dipeptides item.
Connect peptide: Boc-Tyr 1
HOBt 1.15(1.0-1.15)
DCCI 1.15(1.0-1.15)
Method is same to be connect under dipeptides item,
5, connect hexapeptide: protecting group, washing, neutralization: method is same to be connect under dipeptides item.
Method is same to be connect under dipeptides item.
6, connect heptapeptide: protecting group, washing, neutralization: method is same to be connect under dipeptides item.
Connect peptide: Boc-Trp 1
HOBt 1.15(1.0-1.15)
DCCI 1.15(1.0-1.15)
Method is same to be connect under dipeptides item.
2.7, connect octapeptide
Base: HCl/iPrOH, CH2Cl2, mercaptoethanol is deprotected, stirring is drained.
Washing neutralizes: method is same to be connect under dipeptides item.
Method is same to be connect under dipeptides item.
2.8, nonapeptide is connect
Deprotection base: method is same to be connect under octapeptide item.
Washing neutralizes: method is same to be connect under dipeptides item.
Connect peptide: p-D-Glu 1
HOAt 1.15(1.0-1.15)
DIC 1.15(1.0-1.15)
Method is same to be connect under dipeptides item.
Washing: CH2Cl2 washs 1 time, DMF washing 2 times, CH2Cl2 washing 2 times, drains.
It is dry: to set in P2O5 drying box and dry to constant weight, obtain nonapeptide resin.
Specifically, the preparation method of this patent has the advantages that following prominent:
1) when preparing Boc-Pro- resin, Boc-Pro-OH is no longer excessively used, and calculates mole of resin for crude product synthesis Dosage reaches twice of Boc-Pro-OH mole,
2) degree of substitution of BOC-Pro in BOC-Pro- resin is reduced to 0.5mmol/ grams or so, so that solid phase be avoided to connect peptide Later period because the extension of peptide chain causes steric hindrance to become larger, causes peptide bond to form difficulty.
3) solid phase connects D- amino acid in peptide and uses efficient condensing agent HOAt and DlC, and l-amino acid uses HOBt and DCCI, Except L-Histidine and Serine, to reduce cost.
4) pre-activate is first carried out in ice bath, filter out the by-products such as N- acylureas again in -5 DEG C or less progress condensation reactions, Reduce side reaction.
5) 2 to 9 peptides are in de- BOC, and in deprotection reagent, mercaptoethanol capturing agent is added, and reduce to the greatest extent secondary anti- It answers.
6) every time after peptide reaction, dehydrated alcohol and methanol washing is not used, is reduced in washing process because of resin Shrink bring adverse effect.
7) gained nonapeptide resin is 0.5 hour dry or more not in 50 DEG C of baking ovens, avoids high temperature that peptide is caused to be broken.
8) acetone used carries out pre-freeze to -20 DEG C hereinafter, high temperature precipitations is avoided to cause peptide chain irreversible reaction when aminolysis.
9) crude product of higher peptide content and purity can be obtained using above-mentioned steps, synthesis yield reaches 60% or more.Crude product is adopted Purified with HPLC, obtains the intermediate of high-purity.Intermediate is concentrated again, filters, is lyophilized and obtains (the 5-oxo-D- of high-purity Proline)]-leuprorelin acetate, purity >=98.5%.
Crude product purity refers in wavelength 220nm ratio shared by purpose peptide in sample, may contain missing sequence in sample Column, short-movie section, the incomplete sequence of deprotection etc..Crude product purity is to be pressed at the maximum absorption band 220nm of peptide by HPLC method It is general in peptide also to contain moisture and salinity obtained from area normalization method analysis, but the purity of peptide does not count them.
Peptide content refers to all peptides shared percentage in the sample, includes all peptide and moisture, salinity etc. in sample, The content of peptide is analyzed to obtain by external standard method by HPLC method with calculated by peak area.
Therefore, crude product purity and crude product peptide content are the important indicator of crude product quality obtained by evaluation experimental.
Specific embodiment
Embodiment 1Preparation
1.1 Boc-Pro-Cs preparation: weighing Boc-Pro, be dissolved in methanol, feeds intake, weighs by Cs2CO3 excessive 95% Cs2CO3 is dissolved in purified water.Cs2CO3 solution is slowly added into Boc-Pro solution, is sufficiently reacted, at this time Boc-Pro-Cs Solution answers colorless and transparent, pH 7.0-7.5.Solvent is boiled off with water vacuum-concentrcted in 40~50 DEG C of water-baths, obtains solids.Add Enter methanol, evaporated under reduced pressure is repeated twice, and is then dried under vacuum to constant weight in P2O5 drier, and it is dry to obtain Boc-Pro-Cs Product.
1.2Preparation: according toChlorine substitution amount calculate, weigh 2 times of monomer molar amountsBoc-Pro-Cs adds DMF, pours into three-necked flask after being completely dissolved, then swung with DMF and wash eggplant type flask, is incorporated to tree In rouge.45 DEG C of stirring in water bath are reacted 48 hours, are filtered, and resin is washed 3 times with DMF, then are washed to no Cl- with purifying and are reacted (detection of AgNO3 reagent), is finally washed 4 times with dehydrated alcohol, methanol is washed 2 times.40~50 DEG C of drying in baking oven are placed in, then are placed in It is dried under vacuum to constant weight in P2O5 drier, obtains zero P dry product of Boc-Pro-.The substitution equivalents of Boc-Pro is 0.55mmol/ G, reaction yield 94%.
Embodiment 2 connects peptide (by taking 300mmol input amount as an example)
2.1, dipeptides is connect
Weigh 300mmol'sIt sets in 10L reaction kettle, CH2Cl2 is washed 2~3 times, is drained.
It deprotects base: 9-10N HCl/iPrOH780ml+CH2Cl2 624ml+ mercaptoethanol 156ml is added, stir 40 points Clock is drained.Washing: CH2Cl2 is washed 1 time, is drained；DMF is washed 1 time, is drained.
It neutralizes: triethylamine/CH2Cl2 (10/90) is added and washs 1 time, drains.
Washing: DMF is washed 1 time, is drained；CH2Cl2 washs 3 times to being in neutrality, and drains.
Connect peptide: 279.0 grams of Boc-ArgHCl (H2O) (295.92 grams) (900mmol)
140.0 grams of HOBt (1035mmol)
213 grams of DCCI (1035mmol)
Amino acid monomer；HOBt and DCCI, is dissolved with 600mlDMF respectively, first by the lysate of amino acid monomer and HOBt Lysate mixing, ice bath stirring react 30 minutes, then add DCCI lysate, continue after being stirred to react 30 minutes, mistake Filter, gained clear liquid pour into resin, close plug bottleneck, -22 DEG C of reaction 3h.Solvent is drained, with CH2Cl2 washing 1 time, DMF washing 2 Secondary, CH2Cl2 is washed 1 time, is drained.
2.2, tripeptides is connect
Deprotection base, washing, neutralization: method is same to be connect under dipeptides item.
Connect peptide: Boc-Leu (H2O) 208.8 (225) grams (900mmol)
140.0 grams of HOBt (1035mmol)
213 grams of DCCI (1035mmol), method is same to be connect under dipeptides item.
2.3, tetrapeptide is connect
Deprotection base, washing, neutralization: method is same to be connect under dipeptides item.
Connect peptide: 208.8 grams of Boc-D-Leu (900mmol)
140.0 grams of HOAt (1035mmol)
130.4 grams of DIC (1035mmol), method is same to be connect under dipeptides item.
2.4, pentapeptide is connect
Deprotection base, washing, neutralization: method is same to be connect under dipeptides item.
Connect peptide: 253.44 grams of Boc-Tyr (900mmol)
140.0 grams of HOBt (1035mmol)
213 grams of DCCI (1035mmol), method is same to be connect under dipeptides item.
2.5, hexapeptide is connect
Deprotection base, washing, neutralization: method is same to be connect under dipeptides item.
But
Connect peptide: 235.44 grams of Boc-Ser (900mmol)
140.0 grams of HOAt (1035mmol)
130.4 grams of DIC (1035mmol), method is same to be connect under dipeptides item.
2.6, heptapeptide is connect
Deprotection base, washing, neutralization: method is same to be connect under dipeptides item.
Connect peptide: 274.44 grams of Boc-Trp (900mmol)
140.0 grams of HOBt (1035mmol)
213 grams of DCCI (1035mmol), method is same to be connect under dipeptides item.
7, octapeptide is connect
Base: 9-10N HCl/iPrOH780ml+CH2Cl2 624ml+ mercaptoethanol 156ml is deprotected, is stirred 60 minutes, It drains.Washing neutralizes: method is same to be connect under dipeptides item.
Tos
Connect peptide: 737.2 grams of Boc-His (900mmol)
140.0 grams of HOAt (1035mmol)
130.4 grams of DIC (1035mmol), method is same to be connect under dipeptides item
8, nonapeptide is connect
Deprotection base: method is same to be connect under octapeptide item.
Washing neutralizes: method is same to be connect under dipeptides item.
Connect peptide: 115.44 grams of p-D-Glu (900mmol)
140.0 grams of HOAt (1035mmol)
130.4 grams of DIC (1035mmol), method is same to be connect under dipeptides item.
Washing: CH2Cl2 washs 1 time, DMF washing 2 times, CH2Cl2 washing 2 times, drains.
It is dry: to set in P2O5 drying box and dry to constant weight, obtain nonapeptide resin.
3 aminolysis of embodiment
3.1, aminolysis: above-mentioned dried nonapeptide resin is set respectively in 4 3L bottle,suctions, every bottle of addition about 750ml without Water methanol, each addition ethylamine about 1125ml are closed.In rocked at room temperature 24 hours, cooling lower opening plug, filtering, resin use Methanol washs 4 times, merges diafiltration liquid.
3.2 concentrations: 40~45 DEG C of diafiltration liquid are concentrated under reduced pressure (vacuum degree is -0.08~-0.1Mpa) to doing, and methanol is added to dissolve (vacuum degree is -0.08~-0.1Mpa) is concentrated under reduced pressure again to doing, repeatedly three times, at foam-like, obtains concentrate I.
3.3, turn acetate: concentrate I weighing is dissolved, -22 DEG C of acetone precipitations of 50 times of volumes, mistake with 5 times of volumes methanols Filter, 5 times of 50% acetate dissolutions of volume of filter cake, 40~45 DEG C are concentrated under reduced pressure (vacuum degree for -0.08~-0.1Mpa) as adding (vacuum degree is -0.08~-0.1Mpa) is concentrated under reduced pressure to doing, repeatedly at least three times, until obtaining at foam-like in methanol dissolution again Concentrate II.
3.4, precipitate: concentrate II weighing is dissolved, -22 DEG C of acetone precipitations of 50 times of volumes, filtering with 10 times of volumes methanols, With acetone washing 1 time, ether washs 3 times filter cake, drains.
3.5 vacuum drying: filter cake vacuum drying obtains crude product.Must be 320 grams of crude product, peptide content 70%, purity are as follows: 81.3%. Synthesis yield is 62%.
Study exploring experiments example
Embodiment 4 gropes the research of BOC-Pro degree of substitution (mmol/g)
Using the BOC-Pro resin of different degree of substitution, embodiment 2,3 is repeated several times, summary obtains the experiment knot of following table 1 Fruit:
Table 1
Conclusion: can be seen that by 1 result of table, when BOC-Pro resin degree of substitution is 0.55mmol/g, the purity and peptide of crude product Content is optimal；When degree of substitution is reduced to 0.55mmol/g or less, experimental result is poor, does not meet expection；Work as substitution When degree is higher than 0.55mmol/g, experimental result is general.Preferably, BOC-Pro resin degree of substitution is 0.55-1.1mmol/g；It is more excellent Selection of land, resin degree of substitution are 0.55-0.70mmol/g；Most preferably, resin degree of substitution is 0.55mmol/g.
Embodiment 5 gropes the research of deprotection reagent
Using different deprotection reagents, embodiment 2,3 is repeated several times, summary obtains the experimental result of following table 2:
Table 2
Deprotection reagent Addition manner Crude yield Crude product purity Crude product peptide content
Mercaptoethanol 2-9 peptide all adds 62 80.3 70
Mercaptoethanol The addition of 8-9 peptide 27 76 52
Mercaptoethanol The addition of 5-9 peptide 35 80 56
Mercaptoethanol The addition of 2-6 peptide 10 54 37
Ethyl mercaptan 2-9 peptide all adds 27 72 45
Ethyl mercaptan The addition of 2-6 peptide 8 50 32
Conclusion: can be seen that by 2 result of table, can be significantly when 2-9 peptide all uses mercaptoethanol as deprotection reagent Improve the crude yield and crude product peptide content of this experiment in ground.
Embodiment 6 gropes the research of deprotection reagent
Using mercaptoethanol (liquor capacity percentage) dosage, embodiment 2,3 is repeated several times, summary obtains following table 3 Experimental result:
Table 3
Mercaptoethanol dosage Crude yield Crude product purity Crude product peptide content
2.5% 47 70 50
5% 58 77 62
10% 62 80.3 70
20% 62 80.2 69
30% 62 80.2 69
Conclusion: can be seen that by 3 result of table, and mercaptoethanol dosage and experiment obtain the quality of crude product, proportional.It improves The dosage of thioglycol can improve yield, purity and the peptide content of crude product.But works as thioglycol to improve to after 10%, test As a result it is in more constant state.It is therefore preferred that the dosage of this experiment thioglycol is 10% or more；It is highly preferred that sulphur The dosage of base ethyl alcohol is 10%.
Embodiment 7 gropes the research of acetone temperature
Using acetone temperature, embodiment 2,3 is repeated several times, summary obtains the experimental result of following table 4:
Table 4
Conclusion: can be seen that by 4 result of table, and acetone temperature and experiment obtain the quality of crude product, inversely；That is warm acetone Degree is lower, and experimental result is better.When acetone temperature is lower than -10 DEG C, experimental result is preferable；When acetone temperature is down to -22 DEG C, Experimental result is optimal；But when acetone temperature continues decline, experimental result is without significant change.
Therefore, preferably, the temperature of acetone is less than or equal to -10 DEG C for this experiment；It is highly preferred that the temperature of acetone is low In or equal to -22 DEG C；Most preferably, the temperature of acetone is -22 DEG C.
The research of the temperature of 8 condensation reaction of embodiment is groped
It filters out the by-products such as N- acylureas to be condensed at a certain temperature again, embodiment 2,3 is repeated several times, summarizes To the experimental result of following table 5:
Table 5
Condensation temp Crude yield Crude product purity Crude product peptide content
15℃ 15 52 31
10℃ 23 68 40
5℃ 25 70 45
0℃ 30 75 54
-10℃ 40 80 61
-20℃ 62 81 70
-30℃ 60 82 69
Conclusion: can be seen that by the experimental result of table 5, filters out the by-products such as N- acylureas and contracts at a certain temperature again It closes, the lower temperature the better, but after temperature drops to -20 DEG C, experimental result is without significant change.It is therefore preferable that being -20 DEG C.

Claims (7)

1. a kind of prepare (5-oxo-D-Proline)] method of-leuprorelin acetate:
(1) solid phase connects peptide: by preparing raw material, connecing dipeptides, connect tripeptides, connect tetrapeptide, connect pentapeptide, connect hexapeptide, connect heptapeptide, connect octapeptide With the step of connecing nonapeptide, obtain Leuprorelin nonapeptide resin, wherein connect dipeptides to include the step of connecing nonapeptide deprotection base； It then washs, neutralizes, washs again, connecing peptide and detection free ammonia；
Wherein, the preparation raw material include: byResin is with anhydrous CH2Cl2Washing, stirring, is drained；DescribedResin replaces equivalents to be 0.4-0.7mmol/g；
(2) crude product aminolysis: is dissociated from above-mentioned Leuprorelin nonapeptide resin by ethylamine；
(3) HPLC is purified: obtaining leuprorelin acetate intermediate purification product through HPLC preparation column purification；
(4) it is concentrated；
(5) it filters, be lyophilized.
2. preparation method according to claim 1, it is characterised in that described in step (1)Resin takes It is 0.55-0.7mmol/g for equivalents.
3. preparation method according to claim 1, it is characterised in that in step (1), connect in peptide, connect tetrapeptide and nonapeptide For D amino acid, other be L amino acid.
4. preparation method according to claim 3, it is characterised in that in step (1), the D- amino acid is using efficient Condensing agent HOAt and DlC, the l-amino acid use HOBt and DCCI.
5. the preparation method according to claim 4, it is characterised in that in step (1), connect dipeptides to when connecing nonapeptide, it is described to go Protecting group includes: that HCl/iPrOH solution, the CH that concentration is 9~10N is added in the raw material of preparation2Cl2, mercaptoethanol three it is mixed It closes in liquid, stirring is drained, wherein HCl/iPrOH solution, CH2Cl2, thioglycol three volume ratio be 5: 4: 1.
6. preparation method according to claim 1, it is characterised in that in step (1), after peptide reaction, do not use Dehydrated alcohol and methanol washing.
7. preparation method according to claim 1, it is characterised in that in step (2), when aminolysis, acetone used carried out pre-freeze To -20 DEG C or less.
CN201910094532.9A 2019-01-27 2019-01-27 A kind of preparation method of (5-oxo-D-Proline)-leuprorelin acetate Pending CN109912691A (en)

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Application publication date: 20190621